The immune response consists of a cellular response and a humoral response. The cellular response is mediated largely by T lymphocytes (alternatively and equivalently referred to herein as T cells), while the humoral response is mediated by B lymphocytes (alternatively and equivalently referred to herein as B cells).
B cells produce and secrete antibodies in response to the presentation of antigen and MHC class II molecules on the surface of antigen presenting cells. Antigen presentation initiates B cell activation with the engagement of the B cell receptor (BCR) at the cell's surface. Following engagement, the BCR relays signals that are propagated through the cell's interior via signal transduction pathways. These signals lead to changes in B cell gene expression, physiology, and function which underlie B cell activation.
T cells produce costimulatory molecules, including cytokines, that augment antibody production by B cells during the humoral immune response. T-cell produced cytokines also play a role in modulating the activity of T cells themselves. Many T cells also act directly to engulf and destroy cells or agents that they recognize by virtue of the cell surface receptors they possess. The engagement of cell surface receptors on T cells results in the propagation of intracellular signals which provoke changes in T cell gene expression, physiology, and function, which underlie the cellular immune response.
Antigen recognition alone is usually not sufficient to initiate a complete effector T or B cell response. The generation of many B-cell responses is dependent upon an interaction of B cells with CD4+ helper T-cells directed against the same antigen. These helper T-cells express CD40L (CD154) which binds to the cell surface receptor, CD40, on resting B cells. This interaction provides a critical activation signal to B cells. Mutations in the CD40L lead to the X-linked immunodeficiency disorder hyper-IgM syndrome, which is characterized by low levels of IgA and IgG, normal to elevated levels of IgM. absence of germinal center formation, and decreased immune response. In addition, transgenic mice lacking CD40 exhibit reduced graft rejection. (Zanelli et al., Nature Medicine, 6: 629–630, 2000; Schonbeck et al., Cell Mol Life Sci, 58:4–43, 2001).
Non-lymphocyte leukocytes and platelets are also activated by surface receptor engagement in immune response and in response to injury. For example, mast cells and basophils are activated by binding of antigen to surface IgE, while platelets are activated by the binding of thrombin to its receptor.
Intercellular communication between different types of lymphocytes, as well as between lymphocytes and non-lymphocytes in the normally functioning immune system is well known. Much of this communication is mediated by cytokines and their cognate receptors. Cytokine-induced signals begin at the cell surface with a cytokine receptor and are transmitted intracellularly via signal transduction pathways. Many types of cells produce cytokines, and cytokines can induce a variety of responses in a variety of cell types, including leukocytes. The response to a cytokine can be context-dependent as well as cell type specific.
The dysregulation of intercellular communication can perturb leukocyte activity and the regulation of immune responses. Such dysregulation is believed to underlie certain autoimmune disease states, hyper-immune states, and immune-compromised states. Such dysfunction may be cell autonomous or non-cell autonomous with respect to lymphocytes.
The activation of specific signaling pathways in leukocytes determines the quality, magnitude, and duration of immune responses. In response to transplantation, in acute and chronic inflammatory diseases, and in autoimmune responses, it is these pathways that are responsible for the induction, maintenance and exacerbation of undesirable leukocyte responses. Identification of these signaling pathways is desirable in order to provide diagnostic and prognostic tools, as well as therapeutic targets for modulating leukocyte function in a variety of disorders or altered physiological states. In addition, the ability to modulate these pathways and suppress normal immune responses is often desirable, for example in transplantation.
While the extracellular domains and cognate ligands of lymphocyte receptors vary widely, many receptors have similar intracellular domains (such as the “immunoreceptor tyrosine-based activation motif” (ITAM)), and associate with common intracellular signaling molecules.
Tyrosine kinase activation is a critical event in the propagation of intracellular signals by many receptors on lymphocytes, including antigen receptors on B and T cells (for a review see Turner et al, Immunology Today, 21:148–154, 2000, incorporated herein in its entirety by reference)
With regard to the B cell antigen receptor, the BCR is rapidly phosphorylated on tyrosine residues following engagement of the receptor by antigen or other crosslinking agents. This tyrosine phosphorylation leads to associations with several SH2-containing signaling proteins. SH2-containing proteins are known to bind to phosphorylated tyrosine residues in the context of specific amino acid sequences.
Many non-receptor tyrosine kinases have been shown to interact with tyrosine phosphorylated receptors in lymphocytes, including the antigen receptors of B and T cells. These non-receptor tyrosine kinases include members of the src family and the Bruton's tyrosine kinase (BTK) family. Importantly, many of these genes are associated with oncogenesis.
A structurally distinct group of non-receptor tyrosine kinases that associate with tyrosine phosphorylated receptors in lymphocytes are the “SYK” proteins (referred to herein as SYK). SYK is a 72 kilodalton cytoplasmic protein tyrosine kinase that is expressed in a variety of cells of the haematopoietic lineage, including B and T cells. SYK is activated in B cells by aggregation of the B cell antigen receptor (BCR) (Hutchcroft et al., JBC 267:8613–8619, 1992), in T cells by cross-linking the T-cell antigen receptor (TCR) (Chan, A., et al., J. Immunol., 152:4758–4766, 1994; Couture, C., et al., Proc. Natl. Acad. Sci. U.S.A., 91:5301–5305, 1994), in mast cells by aggregation of FcεRI receptors (Hutchcroft et al., PNAS 89:9107–9111, 1992), in platelets by thrombin (Taniguchi, T., et al., J. Biol. Chem 268:2277–2279, 1993) or integrin ligation (Clark, E. A., et al., J. Biol. Chem., 269:28859–28864, 1994), in monocytes by cross-linking FcγRI ad FcγRII receptors (Agarwal, A., et al., J. Biol. Chem., 268:15900–15905, 1993; Kiener, P. A., et al., J. Biol. Chem., 268:24442–24448, 1993), in macrophages by engagement of the FcγRIIIA receptor (Darby, C., et al., J. immunol., 152:5429–5437, 1994), in granulocytes in response to granulocyte stimulating factor, and in peripheral blood lymphocytes by interleukin-2. SYK contains two tandem SH2 domains and multiple tyrosines that when phosphorylated can serve as binding sites for additional signaling proteins including phospholipase C-γ, VAV, and CBL (Junghans, Immunol. Today, 20:401–406, 1999; Sklar et al., Cytometry, 3:161–165, 1982; Robins et al., J. Immunol. Methods, 90:165–172, 1986). SYK is tyrosine phosphorylated in B cells activated by BCR engagement, and is essential for the development and function of B cells. Activation of the BCR (used herein interchangeably with “engagement of the BCR”) at different developmental stages evokes different cellular responses. In immature B cells, stimulation of the newly formed surface immunoglobulin leads to cell death or rearrangement of light chain genes (MacLennan, Curr. Opin. Immunol. 10:22–225, 1998). In mature B cells, BCR engagement leads to proliferation and differentiation into antibody-producing cells or memory B cells (MacLennan, Curr. Opin. Immunol. 10:22–225, 1998). In addition, it is believed that stimulation of the immunoglobulin pathway is required for immature B cells to differentiate into mature, recirculating follicular B cells. Importantly, B cell maturation and humoral immunity are compromised in SYK deficient mice (Turner et al., Nature, 378:298–302, 1995), underscoring the importance of SYK-mediated signal transduction in B and T cells.
The activity of protein tyrosine kinases and other signaling proteins is generally tightly regulated in normal cells. One method of controlling signaling protein activity involves conjugation of ubiquitin or ubiquitin-like proteins to signaling proteins.
Ubiquitin is a 76-amino acid polypeptide that is highly conserved in eukaryotes. Several ubiquitin coding loci identified in yeast are differentially expressed in cells during exponential growth, stationary phase, and during stress such as high temperature or starvation [Ozkaynak et al. EMBO J. 6(5):1429–1439 (1987)]. In one aspect, ubiquitin mediates selective proteolysis by conjugating to intracellular proteins, thereby targeting them to the proteosome where they are cleaved adjacent to the C-terminal of the ubiquitin moiety. Conjugation of ubiquitin to a target protein may also result in an alteration in the subcellular localization or activity of the signaling protein without proteolytic degradation (for example see Depraetere, Nat. Cell Biol., 3:E181).
Such modifications of target proteins are reversible. The level of target protein conjugation is negatively influenced by the action of peptidases with activity specifically directed at ubiquitin. The ubiquitin-specific proteases comprise a family of proteins which have both proteolytic ability and the ability to deubiquitinate the ubiquitin-protein conjugate [Tobias et al., J. Biol. Chem. 266(18):12021–12028 (1991); Baker et al., J. Biol. Chem. 267(32):23364–23375 (1992); Xiao et al., Yeast 10(11):1497–1502 (1994); Baek et al., J. Biol. Chem. 272(41):25560–25565 (1997)]. Deubiquitinating enzymes are able to remove ubiquitin from substrate proteins, thereby interrupting their transport to the proteosome for destruction. A very large number of deubiquitinating enzymes are known to exist, which raises the possibility that individual enzymes may recognize distinct ubiquitin-conjugated substrates. Substrate specificity among deubiquitinating enzymes has been demonstrated previously (Jensen et al., Oncogene 16:1097–1112, 1998; Kahana et al., Mol. Cell. Biol. 19:6608–6620, 1999; Moazed et al., Cell 86:667–677, 1996). Such proteases may remove ubiquitin conjugated to target proteins thereby altering the subcellular localization, activity, and/or proteolytic processing of target proteins.
Coordinated intracellular protein degradation is critical to a vast array of cellular processes, including cytokine signaling in lymphocytes. In addition, it has been suggested that the dysregulation of ubiquitin mediated proteolysis may be involved in the development of cancer in mammals, due to the association of a ubiquitin specific protease with cell cycle regulatory proteins [Xiao et al., supra].
Similarly, conjugation of ubiquitin-like proteins to a target protein often results in the modification of target protein activity and/or subcellular distribution. For example, conjugation of the ubiquitin-like proteins SUMO and NEDD8 to target proteins alters their subcellular localization and stability (Muller et. al., Nat. Rev. Mol. Cell. Biol., 2:202–210, 2001; Yeh et. al., Gene, 248:1–14, 2000).
UBC9 is a ubiquitin-conjugating enzyme which also catalyzes SMT3/SUMO conjugation to target proteins (Schwarz et. al., Proc. Nat'l. Acad. Sci., 95:560–564, 1998). Further, these modifications are also reversible, and proteases may remove ubiquitin-like proteins conjugated to target proteins thereby altering the subcellular localization, activity, and/or proteolytic processing of target proteins (for example see Kim et. al., J. Biol. Chem., 275:14102–14106, 2000).
Compositions that are capable of modulating the conjugation of ubiquitin and ubiquitin-like proteins to signaling proteins are desirable and provide means for modulating signal transduction. Such compositions are desirable for the modulation of leukocyte activation in normal and abnormal immune responses.
In addition to kinases, phosphatases also play an important role in lymphocyte activation. Calcineurin is a calcium-sensitive protein serine/threonine phosphatase comprised of a catalytic and a regulatory subunit. Calcineurin is activated by the binding of Ca2+/calmodulin, and is inhibited by the immunosuppressants FK506 and cyclosporin A. Binding of cyclosporin to calcineurin blocks substrate access to the active site of the phosphatase (Liu et al., Cell, 66: 807, 1991).
The activation of calcineurin is an important regulatory step in lymphocyte activation. Calcineurin regulates phosphorylation of the nuclear factor in activated T cells (NFAT) transcription factor, and thereby regulates nuclear import of NFAT and its ability to regulate transcription. The expression of many factors involved in lymphocyte activation, including cytokines (such as IL-2) and cell surface molecules (eg. CD40L) lies downstream of NFAT activation. (see Klee et al., J Biol Chem., 273:13367, 1998; Stankunas et al., Cold Spring Harbor Symposia Quant. Biol., 64: 505–516, 1999).